Opioid receptor dimerisation studied using a functional complementation technique

Pascal, Geraldine (2005) Opioid receptor dimerisation studied using a functional complementation technique. PhD thesis, University of Glasgow.

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Abstract

In this study, mutated versions of hDOR, hMOR and rKOR fused to G11alphaC351 I protein were constructed. Mutations were introduced either in the receptor or in the G protein part of the fusion proteins. A highly conserved glycine was mutated into alanine (G202A) in the G11alphaC351 I protein abolishing GDP/GTP exchange. For the opioid receptors a pair of valines belonging to the 2nd intracellular loop were mutated in glutamic acid and aspartic acid. This double mutation eliminated agonist-induced receptor activation of the G11alphaC351 I protein. However, when the pair of non-functional fusion proteins were co-expressed agonist induced [35S]-GTPgammaS binding was recovered for all homo and heterodimers tested including the MOR/KOR combination, which was previously indicated as being unable to dimerise. A cross-talk between hDOR and beta2-adrenoceptor fusion proteins was also observed but the reconstituted signal was two times weaker compared to the hDOR homodimer. This result suggested that the affinity of hDOR to homodimerise is higher than to heterodimerise with beta2-adrenoceptor. Ligand binding affinity for the different fusion proteins was assessed and a loss of ligand binding affinity was observed for all the fusion proteins incorporating the pair of mutated valines. However, upon co-expression with the corresponding fusion protein containing the G202 A mutation, the wild-type pharmacology seemed to be recovered. In this study the hDOR N-terminal and/or TM1 were also demonstrated as interacting with the full length hDOR as well as self-associating. Consequently, TM1 is a possible interface for hDOR homodimer formation. Co-expression of membrane tethered hDOR TM1 with full length hDOR did not produce agonist-induced [35S]-GTPgammaS binding, suggesting that two full length receptors are necessary to generate a functional signal.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Graeme Milligan
Keywords: Neurosciences
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-71492
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:31
Last Modified: 10 May 2019 14:31
URI: http://theses.gla.ac.uk/id/eprint/71492

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